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Monday, September 2, 2013

Why optogenetics deserves the hype

Optogenetics has
come in for some stick lately, with a number of people criticising the hype that this
technique generates in some quarters. That’s fair enough, I suppose – there
have no doubt been some claims made about what can be accomplished with this
technique that are, at the very least, premature. I’m all for bashing hype (see
The Trouble with Epigenetics 1
and 2,
for example), but criticising the technique for what it’s not good for seems to
be missing the point to me.

To me, optogenetics will revolutionise
neuroscience. It is the tool that will finally let us meaningfully integrate
the cellular with the systems level. Not by itself, of course – we’ll still
need all the electrophysiology and pharmacology and neuroimaging and lesion
studies and model organisms and whatever you’re having yourself. And not
without some teething problems and over-interpretation of early findings, which
will no doubt earn more tongue-lashings from the hype-police. But it will let
us ask questions we have not been able to ask before – the right questions, at
the level of cell types, the fundamental functional units of the nervous system.

Before I go on, a brief primer on how
optogenetics works: this technique takes advantage of a number of proteins found in
various species of algae that respond to light of certain wavelengths by
opening a channel in their cell membrane to allow electrochemical ions (like
sodium or chloride) to flow in or out of the cell. Controlling the flow of such
ions along their fibres is also how neurons conduct electricity. If you take
the gene that encodes the light-sensitive channel from algae and force neurons
to express it, then they will become responsive to light – if you shine a light
on them they will “fire” an electrical signal, or “action potential”.
If you turn the light off, they will stop firing action potentials. And if you
use a different channel protein, you can silence the neurons and stop them
firing action potentials. This gives very tight, reversible control over the
activity patterns of the neurons expressing these channel proteins (called channelrhodopsins).

The trick, and the power of the technique,
comes from the specificity with which you can direct that expression. This is
based on the fact that different types of cells express different sets of
genes. All genes have two main parts – one part is basically the recipe or code
for a particular protein. The other part, which is encoded on a neighbouring
piece of DNA, is the regulatory region
– the instructions for when and where to make that protein and how much to
make. Those two regions can be separated. You can cut out the DNA that makes up
just the regulatory piece of one gene and hook it up to the protein-coding
region for any other gene you like (in this case, a channelrhodopsin protein).
Now you can take that fusion gene and introduce it to cells or transgenically
introduce it to animals, like worms or flies or mice. Such animals will now
express channelrhodopsin only in the cell types directed by the regulatory
piece of DNA you chose. A variety of other molecular methods can also be used
to achieve this goal, including resources based
on binary systems like the Cre-LoxP recombinase system. (Fibre optics can then be used to target light to those
cells in particular brain regions).

So how many different cell types are we
talking about? Going by the kinds of animations common in science fiction
movies, many people apparently think of the inside of the brain as a network of
effectively identical cells, randomly placed in a sponge-like layout, connecting
simply to their nearest neighbours. Nothing could be further from the truth. We
have known since the time of Ramon y Cajal and Golgi that there
are many distinct types of neurons, which are distributed in a highly organised
fashion in different brain regions and interconnected with exquisite
specificity. And when I say many, I mean many hundreds, possibly thousands of
types.

The retina alone has over 60 distinct, recognised neuronal cell types and more subtypes are being defined all the
time. Those 60 cell types are arranged in four or five distinct layers, with
multiple subtypes in each layer. There are at least a dozen parallel pathways across these several layers, processing various aspects of the
visual stimulus – colour, form, direction, motion and many others. If you want
to understand how the retina works – to reverse engineer it – you need to know
what the functions of these cell types are within the context of the circuit in
which they are embedded.

The importance of cell types as functional
classes is blindingly obvious for the retina, but the same principle applies to
any area of the brain. Subsets of cells in any area not only have discrete jobs
to do within that area, making unique contributions to the computations carried
out there, they also often connect in distinct, cell-type-specific, parallel
circuits with other brain areas.

In the cerebral cortex,
different excitatory cell types are arranged into six
obvious layers, but these often have several sublayers. And within each layer,
there are multiple subtypes of excitatory neuron, intermingled. In layer 5, for
example, some neurons project across the corpus callosum to the other
hemisphere, some within the cortex on their own side and others to subcortical
targets. Each of these types contains multiple subclasses carrying information
to distinct targets. That cellular complexity is multiplied by the number of
cortical areas – subcortically-projecting layer 5 neurons in motor cortex are
molecularly distinct from those in visual cortex, for example.

And we haven’t even started on the interneurons.
These are smaller, more locally projecting cells, which are inhibitory – they
put the brakes on excitation in neural circuits. They not only prevent runaway
excitation, but also, crucially, control many aspects of information
processing, such as filtering, gain control and temporal and spatial
integration. In addition, they orchestrate the synchronous firing of ensembles
of excitatory neurons, which in turn is a central mechanism in mediating
communication between brain areas. Just in the hippocampus, there are twenty-some subtypes of interneurons already known, and, again, more are being defined all the
time. Each of these subtypes is distributed in a particular manner, expresses
different kinds of ion channels and neurotransmitter and neuromodulator
receptors and makes specific kinds of synapses on specific subcellular
locations of specific target cells.

We cannot ignore this cellular complexity,
but, until recently, we have had few options for really embracing it. As long
ago as 1979, the central importance of cell types was recognised. Francis Crick had seen the power of molecular genetic techniques in other areas of
biology and knew that, with the right tools in hand, it could be harnessed to
help unlock the mysteries of the brain. His article in Scientific American’s, “Thinking about the Brain”
explicitly described three needed methods for neuroscience to make real
progress: first, a method by which “all the connections to a single neuron
could be stained”; second, a method by which “all neurons of just one
type could be inactivated, leaving the others more or less unaltered”; and, third, a means to
differentially stain each cortical area, “so that we could see exactly how
many there are, how big each one is and exactly how it is connected to other
areas.”

While connectomics on
various scales is addressing the first and third of these, optogenetics
provides the means to accomplish the second. Indeed, it surpasses the
requirement Crick had in mind, by allowing not just inactivation but also
activation, with exquisite temporal control and rapid reversibility. (As it
happens, optogenetics is also a fantastic method for mapping functional
connections between cell types).

Using optogenetics, we can move beyond the
crude methods of lesion studies or stimulation with electrodes inserted into a
particular brain region. These methods are hopelessly confounded by the
intermingling of cell types within the targeted regions. In any given area, it
is typical to find multiple cell types that directly antagonise each other –
lesioning them all or stimulating them all may not reveal the complex functions
and computations carried out by the region in question. Optogenetics simply
provides a much more precise, selective and controllable method to perform
these kinds of investigations.

One example is provided by the circuitry
controlling appetite. The arcuate nucleus
in the hypothalamus is a crucial hub in this signaling, integrating signals
from the periphery, such as leptin
and insulin levels, and passing these signals on to further hypothalamic
regions which mediate feeding behaviours. Lesioning the arcuate nucleus has
little effect on feeding behaviour, however. The reason for that was discovered
once the leptin receptor and other players in this system were cloned and
molecular genetic characterisations revealed two major cell types intermingled
in the arcuate nucleus. These directly antagonise one another and communicate
opposite signals to downstream areas – it is the balance between their
activities which controls behaviour. Several recent
optogenetics studies
have now greatly increased our understanding of this system, mapping
connectivity to specific cell types in downstream target regions, revealing the
hierarchy of their functional relationships and directly demonstrating short-
and longer-term effects on behaviour of activity of these different neuronal
classes.

These experiments are not just elegant and
precise, they are powerful and incisive – they are the right experiments to do
to understand this system because they interrogate the system at the right
level: the distinct cell types that make up the fundamental computational
units.

Another reason I am so excited by
optogenetics is it provides one means to integrate analyses at very different
levels, uniting what have been disparate areas of neuroscience. The
characteristics of individual neurons or specific synaptic connections are
traditionally analysed by molecular and cellular neuroscience and electrophysiology.
The roles of specific neurotransmitters or receptors are probed with
pharmacology. The functions and interactions of brain areas are studied using
field recordings, electroencephalography, neuroimaging, lesions and other
systems neuroscience methods. These approaches have traditionally been carried
out by different people with different skills and different mindsets.

While we may have learned a lot of details
at each level, integrating knowledge across those levels has remained a huge
challenge. As a result, we have had little real understanding of how the
functions of any brain area emerge from the interactions of its component
cells.

Optogenetics provides a method to connect
those levels. By inhibiting or activating entire classes of neurons within a
region and analysing the effects on activity in other cells or regions or the
effects on behaviour of the animal, on a moment-to-moment basis, we can discern
the functions which these cells and circuits have evolved to perform.

And that’s the key, really – evolution has
built the mammalian brain by elaborating on basic plans already present in our
distant ancestors. In simpler organisms it is possible to identify not just types
of cells, but individual neurons – in nematodes, the 302
neurons have all been named. In insects, you can see the equivalent individual
neurons repeated in each segment of the ventral nerve cord. Those nervous
systems function based on the actions of individual neurons and their
interconnections. Mammalian brains function more at the level of ensembles of
neurons, but the basic logic is similar – evolution has built these brains by
expanding the numbers of cells of ancestral types, so that what was once a
single neuron is now a population of neurons of the same “type”. Evolution has
also increased the diversity of subtypes, which are deployed and combined in myriad ways to generate the
incredibly complex circuitry we seek to understand.

That is the reason I argue that cell types
are the fundamental units of the nervous system and why optogenetics is such a
powerful method to help move neuroscience from crude and fragmented approaches
to a united field capable of explaining how the operations of the mind emerge
from the workings of the brain.

Let me add a few notes:

First of all, I don’t have a dog in this
fight. I have no stake in any optogenetic technologies and don’t currently use the
method, though I certainly hope to in the future. I’m simply really excited by
its potential. I don’t get giddy over new techniques very often, but when I saw
Karl Deisseroth
present his team’s work at the first Wiring the Brain meeting in 2009, I was blown away by its potential – along with
the rest of the audience of hard-to-impress neuroscientists.

Second, optogenetics alone is not the
answer to all things – it is a method that is suitable for asking specific
kinds of questions. There are, in addition, numerous conceptually similar
molecular genetic techniques now being used or developed, which greatly expand
our arsenal of tools for monitoring and manipulating patterns of neuronal
activity.

Third, let’s consider a few of the common
and recent critiques of the method:

1.The drivers we are using do not
target real cell types, because they depend on the expression pattern of single
genes, while real cell types are defined in a combinatorial fashion by the
expression of multiple genes. That is absolutely true, but intersectional strategies (which drive expression only where two genes intersect) are
greatly increasing the specificity possible. Also, combining transgenic drivers
with viral systems that can be delivered to specific brain regions can address
many of these issues.

2.We don’t know what stimulation
protocols to use. Just blasting some neurons so they fire like crazy does not
recapitulate the real patterns of firing seen in vivo. Also true, though that
criticism applies to traditional electrical stimulation as well. But molecular
genetic tools designed to monitor and measure these patterns have also been
developed and such patterns can be retransmitted through the sensitive, rapid
and reversible optogenetic drivers.

3.It’s not good for studying neuromodulation – the slow signaling which is so
important for changes in the functions of neural circuits over longer
timeframes. This is just wrong. You just need to target the neuromodulatory
neurons – the ones that release dopamine or serotonin in response to action
potentials that they fire. Many of the most exciting early papers using
optogenetics have taken this approach. In addition, new techniques, like DREADD, have been
developed to directly activate G-protein-coupled receptors in a way that
closely mimics neuromodulatory effects.

4.It can’t replace lesion studies. Yes, it can. Or at least it can provide a crucial complement. Lesion studies are great for studying
the effects of lesions to specific areas (of obvious clinical importance) but
limited for inferring how the functions of those areas are mediated, for the
reasons outlined above.

5.We can’t use it for therapies because we don’t know which brain regions to target.
Well, first off, deep brain stimulation is currently in use for conditions like
obsessive-compulsive disorder and Parkinson’s disease and is showing great
promise for depression. Optogenetic approaches may provide a more sophisticated
method to control neural activity, which is directed to specific cell types
within the target region. This is likely a long way in the future and would
involve the complex issue of transfecting human brain cells with viruses, but
it is clearly a theoretical possibility and an interesting avenue to explore. Secondly,
optogenetics is primarily a research tool – one that we hope will lead us to a
greater understanding of brain circuit function and dysfunction, which, in
turn, will allow us to develop new therapeutic approaches. When people like
Karl Deisseroth talk about its relevance to psychiatric disease, this is what
they mean: “Despite the enormous efforts of
clinicians and researchers, our limited insight into psychiatric disease (the
worldwide-leading cause of years of life lost to death or disability) hinders
the search for cures and contributes to stigmatization. Clearly, we need new
answers in psychiatry.” As quoted and misrepresented here.

Finally, to end on a positive note, here
are a few of my personal favourites from the recent literature where
optogenetics approaches have generated real and novel insights into the
organisation and function of specific brain circuits:

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In your 5th bullet point under critiques, you mention that deep brain stimulation '"is showing great promise for depression". Could you provide a link to a paper or another media source in evidence of this?

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